Methods of treating and preventing breast cancer with s-equol

ABSTRACT

The present invention provides methods and compositions for treating or preventing breast cancer with S-equol. The method and compositions are particularly suited to treating triple-negative breast cancer. The S-equol may be administered alone or in combination with one or more cytotoxic or immunotherapeutic compound or molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/384,417 filed Apr. 15, 2019, which claims priority from U.S.Provisional Application Ser. No. 62/685,392, filed Jun. 15, 2018, whichis hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with U.S. Government support under grant numberCA206529 awarded by the National Institutes of Health and the TexasCancer Research Grant Contract under grant number DP150055 by the CancerPrevention and Research Institute of Texas. The U.S. Government and theTexas State Government have certain rights in this invention.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to methods and compositions for treating orpreventing breast cancer with a pharmaceutically effective amount ofS-equol or a pharmaceutical composition comprising S-equol. In apreferred embodiment, the invention relates to the treatment oftriple-negative breast cancer. In another embodiment, the method relatesto a combination therapy using S-equol and another anti-cancertreatment, such as immunotherapy.

Description of the Related Art

Breast cancer is highly heterogeneous and consists of multiple subtypes.Triple negative breast cancer (TNBC) is a subtype of breast cancer thatlacks the expression of estrogen receptor α (ERα), progesterone receptor(PR), and human epidermal growth factor receptor 2 (HER2). (Cleator etal. (2007); Kang et al. (2008); Chia and Tutt (2007); Diaz et al.(2007); Gonzalez-Angulo (2007); Reis-Filho and Tutt (2008); Irvin andCarey (2008)). While TNBC constitutes approximately 15% of all breastcancers, mortality of patients with TNBC is disproportionately higherthan those with other subtypes of breast cancer. TNBC tends to have morerapid disease progression (Gerson et al. (2008); Dent et al. (2007);Haffty et al. (2006); Kaplan and Malmgren (2008)), yet the “triplenegative” characteristic excludes TNBC patients from the benefit ofstandard hormonal and HER2-targeted therapies (Baum et al. (2002);Davies et al. (2013); Goss et al. (2005); Santen et al. (2009); Shou etal. (2004); Smith et al. (2005)). Despite several prospects on thetherapeutic horizon for TNBC, chemotherapy remains the only standardtreatment for TNBC patients. Those who are resistant to currentchemotherapies suffer from unnecessary toxicity without substantialclinical benefits. Therefore, there is an urgent need to develop saferand more effective treatment for TNBC.

Recent years have witnessed major clinical breakthroughs in cancerimmunotherapies (Mellman et al. (2011); Kaufman et al. (2013); Brower(2015); Topalian et al. (2015)), which include blocking theimmune-suppressive immune checkpoint molecules CTLA-4 (cytotoxicT-lymphocyte-associated protein 4 or CD152 [cluster of differentiation152]), PD-1 (programmed cell death protein 1), and PD-L1 (programmeddeath ligand 1; Hodi et al. (2010); Wolchok et al. (2013); Topalian etal. (2014)). PD-1 is a checkpoint protein on T-cells that serves as an“off switch” to keep T cells from attacking other cells in the body.PD-1 interacts with PD-L1 on tumor cells. When PD-L1 interacts withPD-1, it prevents the T-cells from attacking the tumor cells. PD-1inhibitors are currently being used to treat certain cancers.

A review of cancer immunotherapy using checkpoint blockade has beenpublished as in Ribas and Wolchok, (2018) (see reference list herein),and is incorporated by reference herein in its entirety, particularlyfor the purposes of discussing pathways to be targeted withimmunotherapy and antibodies for use in that checkpoint blockade. Inaddition, methods of rationally selecting cancer vaccine targets basedon a patient's “mutanome,” namely, a set of somatic mutations thatgenerate cancer-specific neoepitopes which can be recognized byautologous T cells as foreign, are discussed in Sahin and Tureci, (2018)(see reference list herein). This publication is also incorporated byreference herein in its entirety for discussing personalized targetingof tumor antigens such that cells of the immune system (e.g., CD4+Thelper cells and CD8+T cytotoxic cells) can be activated to attack tumorcells. See, FIGS. 10 and 11. Both of these types of cancer immunotherapycan be combined with the use of S-equol in accordance with the presentinvention.

CD4⁺ T cells play a key role in the functioning of a healthy immunesystem. They assist B cells to make antibodies, activate the microbekilling capacity of macrophages and recruit other immune cells toinfected or inflamed areas of the body. These activities areorchestrated through their production of various cytokines andchemokines. It has been known for some time that uncommitted CD4+T-cells can differentiate into Th1 or Th2 cells, based on the prevailingpro-inflammatory/anti-inflammatory environment, and that these activatedTh1 and Th2 cells had distinct cytokine production patterns andfunctions. Generally, Th1 cells were associated with the eradication ofintracellular pathogens whereas Th2 cells were heavily involved inresponses against extracellular pathogens and parasites. UncontrolledTh1 responses were implicated in autoimmunity and aberrant Th2 responseswere associated with allergy and asthma development. However, this modeldid not explain the observation that a deficiency in Th1 signalingand/or cytokines still allowed the development of autoimmune diseasessuch as rheumatoid arthritis and multiple sclerosis. More recently(2006) a third subset of CD4 T cells, Th17 cells, which have apro-inflammatory bias was identified. Subsequent research using animalmodels and human studies has demonstrated a key role for Th17 cells inthe immune system's defense against extracellular bacteria and fungi aswell as the development of autoimmune diseases, mediated by thesecretion of IL-17 by these cells. The secretion of IL-23 fromantigen-presenting cells such as dendritic cells, which have beenactivated by the uptake and processing of pathogens, in turn activatesTh17 cells. (Taken from Bitesized Immunology:https://www.immunology.org/public-information/bitesized-immunology/cells/th17-cells.)See, FIG. 11.

CD8⁺ (cytotoxic) T cells, like CD4⁺ Helper T cells, are generated in thethymus and express the T cell receptor. However, rather than the CD4molecule, cytotoxic T cells express a dimeric coreceptor, CD8, usuallycomposed of one CD8α and one CD8β chain. CD8⁺ T cells recognize peptidespresented by MHC Class I molecules, found on all nucleated cells. TheCD8 heterodimer binds to a conserved portion (the α3 region) of MHCClass I during T cell/antigen presenting cell interactions (see FIG.10). CD8⁺ T cells (often called cytotoxic T lymphocytes, or CTLs) arevery important for immune defense against intracellular pathogens,including viruses and bacteria, and for tumor surveillance. When a CD8⁺T cell recognizes its antigen and becomes activated, it has three majormechanisms to kill infected or malignant cells. The first is secretionof cytokines, primarily TNF-α and IFN-γ, which have anti-tumor andanti-microbial effects. The second major function is the production andrelease of cytotoxic granules. These granules, also found in naturalkiller (NK) cells, contain two families of proteins, perforin, andgranzymes. Perforin forms a pore in the membrane of the target cell,similar to the membrane attack complex of complement. This pore allowsthe granzymes also contained in the cytotoxic granules to enter theinfected or malignant cell. Granzymes are serine proteases which cleavethe proteins inside the cell, shutting down the production of viralproteins and ultimately resulting in apoptosis of the target cell. Thecytotoxic granules are released only in the direction of the targetcell, aligned along the immune synapse, to avoid non-specific bystanderdamage to healthy surrounding tissue (see FIG. 10). CD8⁺ T cells areable to release their granules, kill an infected cell, then move to anew target and kill again, often referred to as serial killing. Thethird major function of CD8⁺ T cell destruction of infected cells is viaFas/FasL interactions. Activated CD8⁺ T cells express FasL on the cellsurface, which binds to its receptor, Fas, on the surface of the targetcell. This binding causes the Fas molecules on the surface of the targetcell to trimerize, which pulls together signaling molecules. Thesesignaling molecules result in the activation of the caspase cascade,which also results in apoptosis of the target cell. Because CD8⁺ T cellscan express both molecules, Fas/FasL interactions are a mechanism bywhich CD8+ T cells can kill each other, called fratricide, to eliminateimmune effector cells during the contraction phase at the end of animmune response. In addition to their critical role in immune defenseagainst viruses, intracellular bacteria, and tumors, CD8⁺ T cells canalso contribute to an excessive immune response that leads toimmunopathology, or immune-mediated damage. (Taken from BitesizedImmunology:https://www.immunology.org/public-information/bitesized-immunology/cells/cd8-t-cells.)

With respect to NK cells, the PK136 monoclonal antibody reacts withmouse NK1.1, an antigen expressed by natural killer cells and a subsetof T cells in the NK1.1 mouse strains including C57BL and NZB. Severalcommonly used laboratory mouse strains such as BALB/c, SJL, AKR, CBA,C3H and A do not express the NK1.1 antigen. For detection of NK cells inthese strains the monoclonal antibody DXS 14-5971 is used. Simultaneousstaining of C57BL/6 spleen cells with PK136 and DXS reveals coexpressionof both markers by a majority of cells as well as presence of smallpopulations of DXS+PK136− and DXS-PK136+ cells.

CD45 (lymphocyte common antigen) and its associated molecules have alsobeen shown to be important for T cell activation. CD45 is areceptor-linked protein tyrosine phosphatase that is expressed on allleucocytes, and which plays a crucial role in the function of thesecells. On T cells the extracellular domain of CD45 is expressed inseveral different isoforms, and the particular isoform(s) expresseddepends on the particular subpopulation of cell, their state ofmaturation, and whether or not they have previously been exposed toantigen. It has been established that the expression of CD45 isessential for the activation of T cells via the TCR, and that differentCD45 isoforms display a different ability to support T cell activation.Although the tyrosine phosphatase activity of the intracellular regionof CD45 has been shown to be crucial for supporting signal transductionfrom the TCR, the nature of the ligands for the different isoforms ofCD45 have been elusive. Moreover, the precise mechanism by whichpotential ligands may regulate CD45 function is unclear. Interestingly,in T cells CD45 has been shown to associate with numerous molecules,both membrane associated and intracellular; these include components ofthe TCR-CD3 complex and CD4/CD8. In addition, CD45 is reported toassociate with several intracellular protein tyrosine kinases includingR561ck and p59fyn of the src family, and ZAP-70 of the Syk family, andwith numerous proteins of 29-34 kDa. These CD45-associated molecules mayplay an important role in regulating CD45 tyrosine phosphatase activityand function. However, although the role of some of the CD45-associatedmolecules (e.g. CD45-AP and LPAP) has become better understood in recentyears, the role of others still remains obscure. See, Altin and Sloan,(1997) in the reference list herein.

However, these highly promising immunotherapies are only effective for asubset of cancer patients and are not usually curative. In particular,clinical trials of anti-PD-1 antibodies in TNBC patients demonstrateonly modest efficacy, with objective responses in the range of 10-20%,and an additional 20% of patients experiencing some stabilization ofdisease that would otherwise be rapidly progressive (Nanda et al. (2016)[KEYNOTE-012 (ClinicalTrials.gov identifier: NCT01848834) was amulticenter, nonrandomized phase Ib trial of single-agent pembrolizumabgiven intravenously at 10 mg/kg every 2 weeks to patients with advancedPD-L1-positive (expression in stroma or >1% of tumor cells byimmunohistochemistry) TNBC . . . A single-agent phase II study examininga 200-mg dose given once every 3 weeks (ClinicalTrials.gov identifier:NCT02447003) is ongoing.]; Emens et al. (2015) [Pts received MPDL3280Aat 15 mg/kg, 20 mg/kg or 1200 mg flat dose IV q3w]; Dirix et al. (2015)[Pts received avelumab at 10 mg/kg Q2W until confirmed progression,unacceptable toxicity, or any criterion for withdrawal occurred.]; Rugoet al. (2015) [Pembrolizumab was administered at a dose of 10 mg/kgevery 2 weeks for up to 24 months or until confirmed progression orintolerable toxicity.]; Dawood and Rugo (2016)). There is thus apressing clinical need to identify better therapies to improveindividual responses to immunotherapy.

PD-1 and its ligand PD-L1 are immune checkpoint molecules that dampen Tcell immunity (Lin et al. (2010); Curiel et al. (2003); Brahmer et al.(2012); Pardoll and Drake (2012); Pardoll (2012)). PD-L1 isoverexpressed in tumor cells and some immune cells, including Tregs (Linet al. (2010); Curiel et al. (2003); Pardoll (2012); Topalian et al.2012)). Immunotherapy with anti-PD-1 ((αPD-1) and anti-PD-L1 antibodies(αPD-L1) has proven successful for treating various cancers (Brahmer(2012); Pardoll and Drake (2012); Pardoll (2012)). Early clinical trialsfor TNBC indicate that PD-1 and PD-L1 are valid targets for interventionand toxicities of treatment are mild. In a Phase 1 study of 27 womenwith heavily pretreated, chemotherapy resistant, metastatic TNBC thatexpressed PD-L1, the response rate was 18.4% to pembrolizumab, an αPD-1antibody (Nanda et al. 2016). A similar study of atezolizumab, an αPD-L1antibody, produced a response rate of 19% in 21 evaluable, PD-L1expressing TNBC tumors (Emens et al. (2015); Dawood and Rugo (2016)).Another group reported an 8.6% response rate to avelumab, another αPD-L1antibody, in 168 TNBC tumors unselected for PD-L1 expression (Dirix etal. 2015), indicating that level of PD-L 1 tumor expression may beimportant in mediating tumor response. Some disease stabilization wasseen in this aggressive subtype of breast cancer, in approximately 20%of patients in these 3 trials. Serious toxicities were few and relatedto immune modulation. These clinical data indicate that the PD-1/PD-L1axis can be a therapeutic target specifically in TNBC, likely incombination approaches for better efficacy.

There are two distinct types of estrogen receptors that mediate diversephysiological effects of estrogens on breast cancer cells. The firstreceptor is the ERα receptor which supports estrogen-dependent breasttumor growth. The second is the ERβ receptor which appears to work in anopposite fashion to significantly attenuate the growth of breast tumorcells in preclinical models (Clarke et al. (2003); Deroo and Korach(2006); Deroo and Buensuceso (2010); Honma et al (2008);Katzenellenbogen and Katzenellenbogen (2000); McDonnell and Norris(2002); Murphy and Watson (2006); Shaaban et al (2008). Thus, ERβ can beviewed in as a tumor suppressor gene in breast cancer. ERβ is expressedin about 40% of TNBC cancers, and the therapeutic potentials oftargeting ERβ have not yet been investigated in TNBC. Two recentadvances have now made it feasible to move from the preclinical modelsand into clinical research. The first advance is a newly discoveredmechanism of rallying ERβ's antitumor activity (Harris (2007); Hartmanet al (2009); Heldring et al. (2007); Thomas and Gustafsson (2011)), andthe second is the development of an oral formulation of an ERβ agonist(S-equol) that has already been tested for clinical safety,pharmacodynamics and tolerance in humans (Jackson et al. (2011a & b);Setchell et al. (20005); Setchell (2002); Schwen et al. (2012).

Estrogen receptor (ERβ; nuclear or cytoplasmic) is reported to bepresent in approximately half of TNBC (Marotti et al. (2010); Reese etal. (2014)). Thus, rallying ERβ antitumor activity through ERβ-specificmodification and/or ligand binding represents an excellent therapeuticopportunity for TNBC. However, the therapeutic potentials of targetingERβ have not been extensively exploited, partly due to the paucity inthe knowledge of how to harness its antitumor activity. Aphosphotyrosine residue (Y36) in ERβ, but not ERα, has recently beenidentified that is important for regulating the antitumor activity ofERβ in TNBC cells (Yuan et al. (2014); Yuan et al. (2016)).

When this phosphotyrosine residue (pY36) was purposefully mutated byadding a phenylalanine group (Y36F), activation of ERβ target genes weredecreased in a TNBC cell line (MDA-MB-231). The Y36F mutation alsoobliterated the ability of ERβ to inhibit tumor cell growth in vitro andin vivo. This preclinical research therefore strongly supports theimportance of pY36 in the antitumor activity of ERβ (Yuan et al., 2014).

There is also evidence that pY36 status correlates with survival ofbreast cancer patients. Research has been done using a Prognostic TissueMicroarray (TMA) from the National Cancer Institute (NCI), whichconsists of a large cohort of breast tumor samples with a clinicalfollow-up record. Using a total of 726 readable IHC samples, patientswith pY36-negative tumors were found to have statistically significantshorter disease-free and overall survival than those with pY36-positivetumors. Interestingly, the association with survival was only seen inStage II & III disease, which raises the intriguing possibility thatpY36 activity may have an effect on disease progression from locallyadvanced to metastatic breast cancer. Collectively, pY36 intensityappears to have a stronger correlation with patient outcome than totalERβ, underscoring the clinical importance of this previouslyunappreciated phosphotyrosine switch. See, FIG. 14.

S-equol, an ERβ agonist, was previously shown to increase respiratoryand maximal glycolysis fluxes in rat hippocampal neurons, as well ascytochrome oxidase (COX) activity and COX1 protein levels in brains fromovariectomized mice, and has been studied in human subjects to assessits health impact and safety (Yao et al. (2013); Jenks et al. (2002);Jackson et al. (2011a & b); Usui et al., (2013)).

S-equol can be produced either chemically (i.e., chemical synthesis) orby biotransformation (biosynthesis) through the metabolism of daidzein,an isoflavone found in soy and red clover, by gut bacteria. Thestructure of S-equol is shown below.

Equol has a chiral center and therefore can exist in two enantiomericforms. S-equol, R-equol, racemic equol, and non-racemic mixtures ofequol (collectively “equol”); compositions of equol; anhydrouscrystalline polymorph of equol; processes for the preparation of equol;and methods of using equol are described in U.S. Pat. No. 8,716,497(filed Sep. 10, 2012); U.S. Pat. No. 8,048,913 (filed Sep. 14, 2009);U.S. Pat. No. 7,960,432 (filed Jul. 3, 2008); U.S. Pat. No. 7,396,855(filed Jul. 24, 2003); U.S. Pat. No. 8,263,790 (filed Jun. 1, 2011);U.S. Pat. No. 7,960,573 (filed May 4, 2009); U.S. Pat. No. 7,528,267(filed Aug. 1, 2005); U.S. Pat. No. 8,668,914 (filed Jul. 31, 2009);U.S. Pat. No. 8,580,846 (filed Aug. 18, 2006); U.S. Pat. No. 8,450,364(filed Apr. 9, 2012); and U.S. Pat. No. 8,153,684 (filed Oct. 2, 2009);U.S. Pat. No. 9,408,824 (filed Mar. 5, 2014); and U.S. Pat. No.9,914,718 (filed Oct. 14, 2015); each of which is hereby incorporated byreference in its entirety.

Formulations comprising isoflavones and products derived therefrom havebeen used in the past to treat disease. For example, a mixture of equol,genistein, and daidzein, or a mixture of equol, genistein, daidzein, andIBSO03569 have shown potential for treating or preventingneurodegeneration and Alzheimer's disease. See Zhao et al. (2009); U.S.Pat. No. 8,552,057; Yao et al. (2013) (collectively “Brinton et al.”).S-equol alone has also been described for treating Alzheimer's Disease.See, U.S. application Ser. No. 15/659,114 published as U.S. PatentPublication 2018/0028491, and International Patent PublicationWO/2018/022604, which are hereby incorporated by reference in theirentireties.

However, there remains a need in the art for methods that utilizeS-equol for the treatment of other disease states that have in the pastbeen difficult to treat. In particular, there is a need in the art formethods that utilize S-equol in the treatment of breast cancer, and evenmore particularly where the breast cancer tumors are negative forestrogen receptor α, progesterone receptor and HER-2 receptor(“triple-negative breast cancer”), and thus not treatable with knownagonists and antagonists of those receptors.

SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

The inventors have found that S-equol, preferably pure and isolatedS-equol, can benefit breast cancer patients. In particular, theinventors have found that S-equol in combination with immunotherapy isparticularly effective in treating breast cancer, and in particular,triple-negative breast cancer.

It is therefore an object of the invention to provide the following:

-   -   1. A method for treating or preventing breast cancer, by        administering a pharmaceutically effective amount of a        formulation comprising S-equol to a subject in need thereof.    -   2. The method of Item 1, wherein the subject has been diagnosed        with triple-negative breast cancer.    -   3. The method of Item 1, wherein the formulation comprises        10-200 mg S-equol.    -   4. The method of Item 1, wherein the formulation comprises        50-150 mg S-equol.    -   5. The method of Item 1, wherein the formulation comprises about        50 mg S-equol.    -   6. The method of Item 1, wherein the formulation comprises about        150 mg S-equol.    -   7. The method of Item 1, wherein the formulation is administered        orally, intravenously, intraperitoneally, or subcutaneously.    -   8. The method of Item 1, wherein said subject is a human.    -   9. The method of Item 1, wherein the S-equol is administered in        combination with one or more other cancer treatments.    -   10. The method of Item 9, wherein the S-equol is administered in        combination with an immunotherapeutic agent.    -   11. The method of Item 10, wherein the immunotherapeutic agent        is an antibody.    -   12. The method of Item 11, wherein the antibody is directed to        programmed cell death protein 1 (PD-1).    -   13. The method of Item 12, wherein the antibody is directed to        programmed death ligand 1 (PDL-1).    -   14. The method of Item 12, wherein the antibody is        pembrolizumab.    -   15. The method of Item 13, wherein the antibody is atezolizumab.    -   16. The method of Item 13, wherein the antibody is avelumab.    -   17. The method of Item 1, wherein the formulation is essentially        free of genistein, daidzein, and/or IBSO03569.    -   18. The method of Item 1, wherein genistein, daidzein, and/or        IBSO03569 are not co-administered with S-equol.    -   19. The method of Item 1, wherein the formulation is essentially        free of R-equol.    -   20. The method of Item 1, wherein the S-equol is produced        chemically.    -   21. The method of Item 1, wherein the formulation is        administered once per day.    -   22. The method of Item 1, wherein the formulation is        administered twice per day.    -   23. The method of Item 1, wherein the formulation is        administered three times per day.    -   24. The method of Item 1, wherein the formulation is        administered four times per day.

The invention also relates to compositions comprising S-equol asdescribed for the methods herein. The invention also includes articlesof commerce comprising a composition that comprises a non-racemicmixture of equol, and typically comprises equol consisting essentiallyof S-equol.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1B. Host effect of ERβ signaling on tumor growth. FIG. 1A showsan immunoblot of mouse ERβ in tissues from WT, KO, and KI animals.Ponceau S staining for protein loading. FIG. 1B shows WT and KI femalemice (n=8) were orthotopically injected with syngeneic murine mammarytumor cells (M-Wnt1, 1×10⁴). *p<0.05.

FIGS. 2A-2D. A role of ERβ signaling in immune cells. FIG. 2A shows theschema for the chimera experiments. FIG. 2B shows tumor growth inchimeric mice (WT>WT: n=10, KI>WT: n=4). p=0.0003. FIG. 2C shows totalCD8⁺ T cells from tumors. FIG. 2D shows interferon gamma (IFN-γ)expressing CD8⁺ T cells from tumors.

FIG. 3. A schema for combination therapy.

FIGS. 4A-4C. Effect of S-equol in combination with an anti-PD-1 antibodyon mice challenged with an AT3 mammary tumor cell line. Mice wereadministered anti-PD-1 antibody at 200 μg every 3 days and S-equol at 50mg/kg per day. FIG. 4A shows that separately, S-equol and anti-PD-1antibody reduce tumor volume compared to control, but S-equol incombination with an anti-PD-1 antibody reduces tumor volume compared toS-equol or anti-PD-1 antibody alone over 35 days post-challenge. FIG. 4Bshows that separately, S-equol and anti-PD-1 antibody reduce tumorweight compared to control, but S-equol in combination with an anti-PD-1antibody reduces tumor weight compared to S-equol or anti-PD-1 antibodyalone. FIG. 4C shows tumors from mice treated with vehicle+IgG2a,S-equol+IgG2a, vehicle+anti-PD-1 antibody and S-equol+anti-PD-1 antibodyand that separately, S-equol and anti-PD-1 antibody reduce tumor sizecompared to control, but S-equol in combination with an anti-PD-1antibody reduces tumor size compared to S-equol or anti-PD-1 antibodyalone.

FIGS. 5A-5D. Analysis of tumor-infiltrating lymphocytes. FIG. 5A showsthat the percentage of CD45⁺CD3⁺ cells increased in mice treated withS-equol plus an anti-PD-1 antibody compared to mice treated with S-equolor anti-PD-1 antibody alone. FIG. 5B shows CD4⁺ cells decreased in micetreated with S-equol plus an anti-PD-1 antibody compared to mice treatedwith S-equol or anti-PD-1 antibody alone. FIG. 5C shows CD8⁺ cellsincreased in mice treated with S-equol plus an anti-PD-1 antibodycompared to mice treated with S-equol or anti-PD-1 antibody alone. FIG.5D shows NK1.1⁺ cells increased in mice treated with S-equol plus ananti-PD-1 antibody compared to mice treated with S-equol or anti-PD-1antibody alone.

FIG. 6. Effect of S-equol in combination with an anti-PD-1 antibody onmice challenged with an E0771 mammary tumor cell line. Mice wereadministered anti-PD-1 antibody at 200 μg every 3 days and S-equol at 50mg/kg per day. FIG. 6 shows that S-equol plus an anti-PD-1 antibodyreduces tumor volume compared to S-equol or anti-PD-1 antibody aloneover 28 days post-challenge.

FIGS. 7A-7B. Effect of S-equol in combination with an anti-PD-1 antibodyon mice challenged with an E0771 mammary tumor cell line. FIG. 7A showstumors from mice treated with vehicle+IgG2a, S-equol+IgG2a,vehicle+anti-PD-1 antibody and S-equol+anti-PD-1 antibody. FIG. 7B showsthat S-equol plus an anti-PD-1 antibody reduces tumor weight compared toS-equol or anti-PD-1 antibody alone.

FIG. 8. S-equol inhibits tumor growth in vivo. A pY36-specificphosphorylation signal was enhanced by the ERα/ERβ common agonist17-β-estradiol and two ERβ-specific agonists diarylpropionitrile (DPN)and S-equol in MDA-MB-231 cells. S-equol treatment inhibited MDA-MB-231cell-derived xenograft tumor growth (n=5). Expression of Ki67 inxenograft tumors. ERβ-pY36 signal in vehicle- and S-equol-treatedxenograft tumor samples. *p,0.05, **p<0.01. See, Yuan et al. (2016)listed below, which is incorporated by reference herein for allpurposes.

FIG. 9. Generalized protocol for treatment of patients with S-equol.

FIG. 10. Schematic depiction of the action of CD8⁺ cells.

FIG. 11. Schematic depiction of the action of CD4⁺ cells.

FIG. 12. Results from pilot clinical study (Example 7 below).

FIG. 13. Schematic for human clinical trial with S-equol.

FIGS. 14A-14B. Association of pY36 with disease outcomes in breastcancer.

FIG. 14A shows the Kaplan-Meier estimate of disease-free and overallsurvival in Stages II-III specimens correlating with IHC intensity ofpY36-ERβ. FIG. 14B shows the Kaplan-Meier estimate of disease-free andoverall survival in Stages II-III specimens correlating with IHCintensity of (A) and total ERβ IHC.

FIGS. 15A-15B. S-equol reduces the growth of TNBC cells in a xenograftmouse model. FIG. 15A shows Ki-67 positivity in tumors of control andS-equol treated mice. FIG. 15B shows images of Ki-67immunohistochemistry (TIC; ×40).

FIGS. 16A-16C. S-equol and/or α-PD-1 treatment with EMT6 mammary tumors.Mice were injected with 3×10⁵ EMT6 cells. α-PD-1 was administered at 200μg/mouse, intraperitoneally every 3 days and S-equol was administered at50 mg/kg/day by oral gavage daily. FIG. 16A shows tumor volume over 28days post-tumor challenge with either vehicle plus α-IgG2a (blue line),S-equol plus α-IgG2a (red line), vehicle plus α-PD-1 (purple line) andS-equol plus α-PD-1 (green line). FIG. 16B shows EMT6 tumor weightpost-tumor challenge with either vehicle plus α-IgG2a (blue dots),S-equol plus α-IgG2a (red dots), vehicle plus α-PD-1 (purple dots) andS-equol plus α-PD-1 (greendots). FIG. 16C shows the size of tumorspost-tumor challenge with either vehicle plus α-IgG2a (top row), S-equolplus α-IgG2a (second row from top), vehicle plus α-PD-1 (third row fromtop) and S-equol plus α-PD-1 (bottom row).

FIGS. 17A-17H. Expression of markers on EMT6 tumor cells aftertreatment. FIG. 17A shows CD45 expression. FIG. 17A shows CD45expression. FIG. 17B shows CD8 expression. FIG. 17C shows CD4expression. FIG. 17D shows NK1.1 expression. FIG. 17E shows CD107aexpression as percent CD3 expression. FIG. 17F shows CD107a expressionas percent CD8 expression. FIG. 17G shows CD107a expression as percentCD4 expression. FIG. 17H shows CD107a expression as percent NK1.1expression.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. Generally, nomenclatures utilized inconnection with, and techniques of, cell and molecular biology andchemistry are those well-known and commonly used in the art. Certainexperimental techniques, not specifically defined, are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. For purposes ofclarity, the following terms are defined below.

“Substantially pure” means about 90% pure, preferably 95% pure, and morepreferably 98% pure of any contaminating proteins.

“Substantially free” means about less than about 10%, preferably lessthan 5%, and more preferably less than 2% of any contaminating proteins.

“Approximately” or “about” means within +/−10%, preferably within +/−5%,more preferably within +/−2% of that which is being measured.

“TNBC tumors” are defined as less than or equal to 5% nuclear stainingof carcinoma cells for ER-alpha and PR and either 0, 1⁺ or 2+ stainingfor HER-2 by IHC. If only fluorescence in situ hybridization (FISH) isperformed for HER-2, it must be less than or equal to 2.0. Tumor cellsthat show distinct nuclear staining of total ER-beta or pY36 (regardlessof cytoplasmic staining) will be scored as positive.

The present invention relates to the prevention and/or treatment ofbreast cancer with S-equol. The inventors have found that S-equol is aparticularly useful treatment for triple-negative breast cancer (TNBC),for which effective immunotherapies are limited.

Dosage amounts and administration schedules for S-equol will depend onwhether the S-equol is being administered prophylactically to a patientat risk of developing breast cancer, or being administered as treatmentfor a patient already diagnosed with breast cancer. A person at risk fordeveloping breast cancer may be a person with a family history of breastcancer, and/or may have one or more mutations in a BRCA1 or BRCA2 gene.In a patient already diagnosed with breast cancer, dosages andadministration schedules may also vary depending on the stage of thecancer (stage I, stage II, stage III, stage IV or stage V), the numberof lymph nodes involved, tumor size and the availability of and/ordecision to co-administer other therapies. Dosages and administrationschedules may also vary depending on various molecular markers presenton or in tumor cells, including, but not limited to ERα, PR, HER2, Ki67and pY36. Diagnostic tests for markers which are prognostic for theaggressiveness and/or likelihood of recurrence of a tumor include theOncotypeDX® Breast Cancer Test, MammaPrint®, PAM-50 ROR®, EndoPredict®and the Breast Cancer Index®. Such markers include, but are not limitedto proliferation genes such as Ki67, STK15, Survivin, CCNB1 (CyclineB1), MYBL2, invasion genes such as MMP11 (Stromolysin 3), CTSL2(Cathepsin L2), HER2 genes GRB2 and HER2, Estrogen genes ER, PGR, BCL2and SCUBE2 and other cancer related genes such as GSTM1, CD68 and BAG1.(Kittaneh et al. (2013); see also, Coates et al. (2015), both of whichare hereby incorporated by reference in their entireties for allpurposes.)

S-equol can be administered one or more times per day at 1-400 mg perdose, more preferably 10-320 mg, more preferably 50-150 mg. Non-limitingexamples include 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 40 mg, 50 mg, 80 mg,100 mg, 150 mg, 160 mg, 200 mg, 250 mg, 300 mg 320 mg, etc. orapproximately or about those doses. The dose may be administered one,two, three or four times per day, preferably twice per day (B.I.D.) Theregimen can be continued indefinitely, or for such time in intervalswhere markers are examined and responsiveness to the dose is determined.Examples of such intervals are two weeks, four weeks, two months, fourmonths, six months, etc. or about or approximately those intervals. Noupper limit, with respect to administration schedule, is required.

The S-equol administered is preferably formulated for oraladministration; however, other routes of administration are alsocontemplated, including rectal, optical, buccal (for examplesublingual), parenteral (for example subcutaneous, intramuscular,intradermal and intravenous) and transdermal administration.

Compositions or formulations according to the present invention cancomprise one or more pharmaceutically-acceptable or industrial standardfillers. The filler must not be deleterious to a subject treated withthe composition. The filler can be solid or a liquid, or both. Thefiller can be formulated with the active S-equol as a unit-dose, forexample a tablet, which can typically contain from about 10% to 80% byweight of S-equol. Compositions can be prepared by any of the well knowntechniques of pharmacy, for example admixing the components, optionallyincluding excipients, diluents (for example water) and auxiliaries asare well known in the pharmaceutical field.

Compositions suitable for oral administration can be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the extract; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchcompositions can be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active S-equol andone or more suitable carriers (which can contain one or more accessoryingredients as noted above). In general the compositions of theinvention are prepared by uniformly and intimately admixing the S-equolwith a liquid or finely divided solid carrier, or both, and then, ifnecessary, shaping the resulting mixture. For example, a tablet can beprepared by comprising or moulding a powder or granules containing theextract, optionally with one or more accessory ingredients. Compressedtablets can be prepared by compressing in a suitable machine, theextracts in the form of a powder or granules optionally mixed with abinder, lubricant, inert diluents, and/or surface active/dispersingagent(s). Moulded tablets can be made by moulding, in a suitablemachine, the powdered compound moistened with an inert liquid binder.

Suitable fillers, such as sugars, for example lactose, saccharose,mannitol or sorbitol, cellulose preparations and/or calcium phosphates,for example tricalcium phosphate or calcium hydrogen phosphate, and alsobinders such as starch pastes using, for example, corn, wheat, rice orpotato starch, gelatin, tragacanth, methylceullose and/orpolyvinylpyrrolidone, and, if desired, disintegrators, such as theabove-mentioned starches, also carboxymethyl starch, cross linkedpolyvinyl pyrrolidone, agar or alginic acid or a salt thereof, such assodium alginate. Excipients can be flow conditioners and lubricants, forexample silicic acid, talc, stearic acid or salts thereof, such asmagnesium or calcium stearate, and/or polyethylene glycol. Dragee coresare provided with suitable, optionally enteric, coatings, there beingused, inter alia, concentrated sugar solutions which can comprise gumarabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titaniumdioxide, or coating solutions in suitable organic solvents or solventmixtures, or, for the preparation of enteric coatings, solutions ofsuitable cellulose preparations, such as microcrystalline cellulose,acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate.Dyes or pigments can be added to the tablets or dragee coatings, forexample for identification purposes or to indicate different doses ofactive ingredients.

Other orally administrable pharmaceutical compositions are dry-filledcapsules made, for example, of gelatin, and soft, sealed capsules madeof gelatin and a plasticiser, such as glycerol or sorbitol. Thedry-filled capsules can comprise the extracts in the form of granules,for example in admixture with fillers, such as lactose, binders, such asstarches, and/or glicants, such as talc or magnesium stearate, and,where appropriate, stabilizers. In soft capsules, the extract ispreferably dissolved or suspended in suitable liquids, such as fattyoils, paraffin oil or liquid polyethylene glycols, to which stabilizerscan also be added.

According to one aspect of the invention, the compositions comprisingS-equol include those described in U.S. Pat. No. 7,960,432 (filed Jul.3, 2008); U.S. Pat. No. 7,396,855 (filed Jul. 24, 2003); and U.S. Pat.No. 9,408,824 (filed Mar. 5, 2014)—the disclosures of each are herebyincorporated by reference in their entireties.

According to another aspect of the invention, S-equol can be preparedchemically (i.e., chemical synthesis) according to the processesdescribed in U.S. Pat. No. 8,716,497 (filed Sep. 10, 2012); U.S. Pat.No. 8,263,790 (filed Jun. 1, 2011); U.S. Pat. No. 7,960,573 (filed May4, 2009); U.S. Pat. No. 7,528,267 (filed Aug. 1, 2005) and U.S. Pat. No.9,914,718 (filed Oct. 14, 2014)—the disclosures of each are herebyincorporated by reference in their entireties. For example, S-equol canbe enantioselectively prepared using an iridium catalyst with a chiralligand. These methods of enantioselectively preparing S-equol areincorporated by reference.

According to another aspect of the invention, S-equol can be a singleanhydrous crystalline polymorph of S-equol, such as the anhydrouscrystalline polymorph of S-equol described in U.S. Pat. No. 9,914,718(application Ser. No. 14/883,617, filed Oct. 14, 2015)—the disclosure ofwhich, including the chemical and physical properties used tocharacterize the anhydrous crystalline polymorph of S-equol, isincorporated by reference in their entireties. For example, theanhydrous crystalline polymorph of S-equol described in U.S. PatentApplication Publication No. 2016/0102070 has the followingcharacteristic X-ray powder diffraction pattern wavenumbers (cm¹): 3433,3023, 3003, 2908, 2844, 1889, 1614, 1594, 1517, 1508, 1469, 1454, 1438,1400, 1361, 1323, 1295, 1276, 1261, 1234, 1213, 1176, 1156, 1116, 1064,1020, 935, 897, 865, 840, 825, 810, 769, 734, 631, 616, 547, 517, 480,and 461. The characterizations of anhydrous crystalline polymorph ofS-equol are incorporated by reference.

S-equol can be administered in combination with one or more additionalcancer treatments, including surgery (breast+/−axilla), cytotoxicchemotherapy, radiation, immunotherapy, cancer vaccines, inhibitors ofcellular pathways (protein or peptide, nucleic acid-based [antisenseoligonucleotides including DNA oligonucleotides, antisense siRNA, shRNA]and/or hormonal (adjuvant endocrine) therapy. Examples of cytotoxicchemotherapy suitable for treating breast cancer include, but are notlimited to anthracyclines such as doxorubicin (Adriamycin®) andepirubicin (Ellence®), taxanes such as paclitaxel (Taxol®) and docetaxel(Taxotere®), 5-fluorouracil (5-FU), capecitabine, cyclophosphamide(Cytoxan®) and carboplatin (Paraplatin®). Examples of adjuvant endocrinetherapy include tamoxifen and aromatase inhibitors such as anastrozole(Arimedix®), exemestane (Aromasin®) and Letrozole (Femara®). Examples ofimmunotherapy include but are not limited to pembrolizumab (Keytruda®,MK-3475), nivolumab (Opdivo®), durvalumab (MEDI4736), tremelimumab,atezolizumab (MPDL3280A), avelumab, trastuzumab, PDR001, and MGD009. Anexample of a known inhibitor of a cellular pathway is a tyrosine kinaseinhibitor such as lapatinib. Appropriate dosages of immunotherapymonoclonal antibodies or other cancer therapeutic are determined basedon the indication, and their determination is well within the skill inthe art. Dosing may be in mg, in mg/kg or mg/m². See, Sachs et al.(2016), which is incorporated by reference herein in its entirety forall purposes, including examples of therapeutic antibodies, smallmolecules and dosages. Pembrolizumab (marketed as Keytruda by Merck) wasthe first mAb approved in the United States targeting PDCD1 (PD-1)receptor. The dosing strategy for this novel immuno-oncology compoundwas driven by understanding of BED. First, an optimally designed,13-patient, within-subject dose escalation study focused on elucidatingthe pharmacokinetic-pharmacodynamic relationship by measuring IL2response over a 2,000-fold dose range of 0.005 to 10 mg/kg (25). The BEDwas estimated to be 2 mg/kg because IL2 stimulation approachedsaturation at exposures consistent with this dose. The BED of 2 mg/kgand maximum administered dose of 10 mg/kg were explored in laterclinical studies. Early clinical response measured by change in tumorsize from baseline was used to perform exposure-response analysisdemonstrating similar antitumor response over the dose range from 2 to10 mg/kg (26). The S-equol and/or additional therapeutic composition maybe administered once, twice, three times or more per day, depending onthe dose, toxicity and indication. Dosing may occur over several days,weeks or months, and may be continuous or intermittent. Typical dosagesfor immunotherapeutics are 50-500 mg/day, more preferably 100-400mg/day, more preferably 150-250 mg/day. In certain embodiments, the dosemay be 150 mg/day or 150 mg twice per day, or 250 mg/day or 250 mg twicea day. In other embodiments the dosage may be given based on patientweight, for instance 0.5-10 mg/kg, more preferably about 1, 2 or 5mg/kg. Dosage may also be given as mg/m², for example 50-500 mg/m², morepreferably 100-300 mg/m², for example about 100, 150, 200, 250 or 300mg/m².

The dosages may be about or greater than the lower end of any of theafore-stated ranges, or about or less than the upper end of any of theafore-stated ranges.

Responsiveness to the S-equol+/−other cancer therapy can be assessed byanalyzing one or more cellular markers, by imaging techniques, bycellular proliferation assays and by direct examination of tumors. Theanalysis of cellular markers can be done with immunohistochemical orimmunocytochemical techniques or by nucleic acid detection, such ashybridization techniques and polymerase chain reaction (PCR). Apreferred biomarker to assess mitotic division and tumor growth is Ki67(Beelen et al. (2012); Urruticoechea et al. (2005)).

The following examples are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

EXAMPLES

The processes of the present invention will be better understood inconnection with the following examples, which are intended as anillustration only and without limiting the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the processes,formulations and/or methods of the invention may be made withoutdeparting from the spirit of the invention and the scope of the appendedclaims.

Example 1

To determine whether the newly identified phosphotyrosine switchregulates ERβ tumor-extrinsic function, a whole-body knock-in (K1) mousemodel (C57BL/6) was established in which the corresponding tyrosineresidue of endogenous mouse ERβ is mutated to phenylalanine (Y-F).Consistent with previously reported ERβ knockout (KO) mice (Krege et al.(1998)), these phosphorylation mutant KI mice had no overt developmentaldefects and were grossly indistinguishable from their WT littermates(unpublished data). Survey of ERβ expression in KO and KI mice indicatedthat the Y-F mutant protein was expressed at levels comparable to WT ERβin multiple tissues, including bone marrow and spleen (FIG. 1A).Syngeneic murine tumor cells of various origins were then transplanted,including the MMTV-Wnt mammary tumor cell line with a basal-like breastcancer profile (Pfefferle et al. (2013)), into WT and KI recipient mice.In all cases, tumor cells grew more robustly in ERβ KI recipient micethan in their syngeneic WT counterparts (FIG. 1B, and data not shown).These data clearly demonstrate that the phosphotyrosine switch isimportant for ERβ tumor-extrinsic antitumor activity in multiple tumortypes.

Example 2

The following experiment sought to delineate further the host ERβsignaling in tumor inhibition based on the role of antitumor immunityand recent clinical advances in cancer immunotherapies (Topalian et al.(2015); Chen and Flies (2013); Sharma and Allison (2015)). A mousechimera experiment was conducted involving a bone marrow transplant. Asillustrated in FIG. 2A, WT recipient mice were first irradiated (10 Gy)to kill endogenous bone marrow cells, followed by transplant with bonemarrow from syngeneic KI or WT donors. After confirming successfulchimerism in KI>WT and WT>WT mice, tumor cells were injected 8 weeksafter bone marrow transplant. Tumor growth was significantly greater inKI>WT chimeras (with KI immune cells) versus WT>WT controls (FIG. 2B).This suggests that KI immune cells poorly controlled tumor growth. Thus,these data link the importance of tumor extrinsic ERβ antitumor activityto the immune response.

Example 3

Next, tumor-infiltrating immune cell populations were analyzed. Totalnumbers of tumor-infiltrating CD4⁺ and CD8⁺ T cells were reduced inKI>WT versus WT>WT mice (FIG. 2C and data not shown), further supportingthe notion that antitumor immunity in KI mice is compromised.Furthermore, the prevalence of IFNγ-producing CD8⁺ (antitumor) cells wassignificantly lower in tumors from KI>WT versus WT>WT mice (FIG. 2D),suggesting compromised cytotoxic potency of CD8⁺ T cells in the absenceof functional ERβ signaling. Additional preliminary data not shown hereindicate that activation of dendritic cells, which prime antitumor Tcells, was also compromised in KI>WT chimeric mice, as evidenced bytheir reduced MHC-II expression. Furthermore, effector T cells were lessactivated (lower CD44/CD62L) and had other reduced effector functions inKI>WT chimeras [e.g., lower tumor necrosis factor alpha (TNF)α,interleukin (IL)-2, and perforin, data not shown]. These data stronglysuggest ERβ-dependent augmentation of antitumor CD8⁺ T cell effectoractivity and improved intra-tumor immune cell accumulation. Therefore,rallying ERβ antitumor activity with clinically safe ERβ agonists suchas S-equol will improve efficacy of existing anticancer immunotherapiesand make them more effective to treat TNBC.

Example 4

The following three murine mammary tumor lines were tested to determinethe effects of a PD-1 inhibitor, S-equol and a combination of the twotherapies on tumor growth: (1) E0771 (B6 background), (2) AT-3 (B6background), and (3) 4T1 (BalbC background). This experimental designincludes four arms (using AT-3 as an example): (1) controls, (2) αPD-1,(3) S-equol, and (4) αPD-1+S-equol (FIG. 3) by injecting into mice 2×10⁵cells subcutaneously into the 4^(th) mammary gland fat pad. Antibodieswere administered (α-PD-1: 200 μg/mice by i.p. injection every 3 daysand S-equol was administered at 50 mg/kg/day by oral gavage. Treatmentbegan 7 day after tumor challenge. Tumor growth trajectories werecompared within treated mice using a repeated measures linear mixedmodel. The primary outcome was the logarithm of the tumor size, and thetest statistic is the treatment×time interaction. Tumors were measuredabout 10 times. The number of recipient mice use used in the studies(n=8 per group) was based on the above study. Results (FIGS. 4-7) fromthis experiment demonstrate the ability of S-equol to boost anticancerimmunotherapy.

Example 5

In addition to measurements of tumor size and weight in each arm,immunophenotyping was performed to gain more mechanistic insight intothe antitumor effects of mono- and combinational therapies. Tumors,spleens, and draining lymph nodes were harvested and weighed.Anti-coagulated blood was collected by cardiac puncture. Tumors wereparaffin-embedded for immunohistochemistry and snap-frozen for mRNA andprotein analysis by Luminex. Flow cytometry was used (forphenotype/functions) and ViCell (for quantification) to analyze immunecells from tumor infiltrates, tumor-draining lymph nodes, and spleens.Analyses include CD3⁺ total T cells and CD4⁺ and CD8⁺ T cell subsets;effector function (e.g., IFN-γ, TNF-α, IL-2, perforin, CD107a);activation (e.g., CD69, CD44, CD62L); and exhaustion (e.g., PD-1, Tim3,Lag3). Antigen-presenting cells (CD11b⁺CD1c⁻ monocyte/macrophages,CD11b⁺CD11c+ dendritic cells), NK1.1⁺ NKp46⁺ NK cells, regulatory Tcells (Treg, CD3⁺CD4⁺CD25^(hi)Foxp3⁺), and myeloid-derived suppressorcells (MDSC, CD11b⁺Gr-1^(hi)) were also analyzed. CD8⁺ T cell receptordiversity were assessed with a commercial kit (AdaptiveBiotechnologies). Local cell proliferation was also tested with Ki67 andBrdU stain.

Example 6

Further work in a preclinical animal model supports the notion of usingS-equol to treat TNBC breast cancer. In immunocompromised mousexenograft experiments, which used MDA-MB-231, a human TNBC breast cancercell line, growth of tumors was suppressed by 60% with S-equol treatmentcompared to control. 5×10⁶ MDA-MB-231 cells were injected orthotopicallyinto mammary gland fat pads of 6 week-old female athymic nude mice(Harlan). When the tumor masses reached 50 to 80 mm³ (about one weekafter the inoculation), the mice were given daily subcutaneousinjections of S-equol (20 or 60 mg/kg per day) or PBS as a vehiclecontrol. Tumor development was followed by caliper measurements alongtwo orthogonal axes: length (L) and width (W) and volume (V) wasestimated by the formula V=[L×(W²)]/2. Xenograft tumors harvested frommice were fixed in 10% neutral-buffered formalin, dehydrated, embeddedin paraffin, and sectioned at 3 μm thickness. Representative tumorsections from vehicle control and S-equol-treated mice were tested forKi-67 expression to assess cell proliferation, and for ERβ pY36.Statistical significance in the experiments was assessed by two-tailedStudent's t test. In all assays, p<0.05 was considered statisticallysignificant. The results of these experiments are shown in FIGS. 8 and15. See, Yuan et al. 2016 listed below, which is incorporated byreference herein for all purposes.

Example 7

A pilot biomarker clinical study was performed in 20 subjects withdocumented TNBC to determine the effect of the ERβ agonist S-equol onKi67 an indicator of tumor cell proliferation. Tumor cell proliferationwas measured by immunohistological staining for Ki-67. See, FIG. 13 forthe outline of the protocol. Ki-67 is encoded by the gene MKI67 gene andis required to maintain individual mitotic chromosomes dispersed in thecytoplasm following nuclear envelope disassembly. Measuring the fractionof Ki-67-positive cells in a tumor is known to be a reliable parameterfor assessing cancer patient prognosis. Subjects with TNBC are known tohave very high levels of Ki67.

Patients enrolled in the study received a history, physical, laboratoryassessment, radiological imaging, and diagnostic biopsies.Immunohistochemistry (IHC) of Ki67 (proliferation marker), total ERβ,and pY36-ERβ (phosphorylated ERβ), B7H1 (also known as PDL-1) and BRCA1(breast cancer 1, early onset) were conducted using core needle biopsytissue samples prior to treatment. Patients were given oral S-equol 50mg, twice daily for approximately two weeks (10-21 days) prior to thescheduled oncologic surgeries or start of primary systemic therapy.Tumor samples were obtained from the surgery or the two-week repeat coreneedle biopsy. Post-treatment IHC evaluation of Ki67, total ERβ,phosphorylated ERβ (pY36) and BRCA1 were obtained. Ki67 reduction is avalidated surrogate marker of both short and long term hormonal therapyefficacy in trials of human breast cancer and was compared pre- andpost-treatment with S-equol. S-equol caused a measurable decrease inKi67, indicating its efficacy in this tumor type. A c-DNA microarrayplatform was also used to discover downstream transcriptional targets ofERβ resulting from activation by S-equol.

Women eligible for the study had newly diagnosed, previously untreated,triple negative breast cancer, with an intact primary breast tumor ofany size. All stages of the disease were eligible. They took S-equolduring the time they underwent standard preoperative evaluation orpre-therapeutic work-up for advanced disease, such as staging proceduresor central line placement. If primary surgery was performed, tissueleftover after standard diagnostic evaluation was used for the two-weekbiomarker assessment. If the patient did not have primary surgery at theend of the two-week S-equol treatment, a second set of 14-gauge coreneedle biopsies was obtained before the start of any standard systemictherapy. See, FIG. 9.

A standard procedure for acquiring core biopsy with a Bard 14-gaugeneedle was utilized for determining TNBC prior to enrollment. Wheneverpossible, the tumor tissues used for IHC analyses were acquired at thesame time as the diagnostic core biopsy. The tissue samples were sentfor staining of total and pY36 ERβ and Ki67, and analyzed. Testing forERα, PR, and HER-2 was performed using validated methods. TNBC tumorswere defined as less than 5% nuclear staining of carcinoma cells for ERαand PR and either 0, 1, or 2+ staining for HER-2 by IHC. Whole-GenomeDASL HT platform from Illumina, which is designed for gene expressionprofiling with extremely low input of RNA (50 pg), was used tointerrogate paired FFPE tissue obtained before and after exposure toS-equol.

Statistical analysis was conducted which showed that ERβ-expressing TNBCresponds to S-equol as manifested by measurable decline in Ki67. Theprimary analysis estimated the geometric mean change of the Ki67expression from baseline to two weeks. This was performed using aone-sample t-test of the pre/post differences in the log-transformeddata, and summarized by the 95% confidence interval. According to theIMPACT trial, the changes in the geometric mean of Ki67 expression aftertwo weeks were −76, −59.5, and −63.9% for the anastrazole, tamoxifen,and combination groups respectively, with a standard deviation ofapproximately 1.0 on the log-scale. This implies effect sizes of 1.0 to1.5 and a power >90% for testing for a decrease in Ki67 expression withtwo-sided α=0.05 and a sample size of at least 45 patients accountingfor variation in accrual and potential drop out. All computations wereperformed with SAS v9.2+(Cary, N.C.) or R v2.15+(Vienna, Austria). 20patients enrolled and completed the study. 17 patients had evaluablepre- and post-treatment samples. Of these 17 evaluable patients, 4patients had greater than 30% reduction in Ki-67, one patient hadgreater than 20% reduction, and 7 patients had 0-20% reduction (5patients had no reduction in Ki-67). See, FIG. 12.

Example 8

A follow-up clinical study enrolls 25 newly diagnosed TNBC patients, andpatients are treated and assessed as discussed in Example 7, except thatthe dose of S-equol is increased to 150 mg twice daily. Treatment withS-equol is for 10-21 days. Ki67 is analyzed as described in Example 7.

REFERENCES

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

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1. A method for treating or preventing breast cancer, comprisingadministering a pharmaceutically effective amount of a formulationcomprising S-equol to a subject in need thereof.
 2. The method of claim1, wherein the subject has been diagnosed with triple-negative breastcancer.
 3. The method of claim 1, wherein the formulation comprises10-200 mg S-equol.
 4. The method of claim 1, wherein the formulationcomprises 50-150 mg S-equol.
 5. The method of claim 1, wherein theformulation comprises about 50 mg S-equol.
 6. The method of claim 1,wherein the formulation comprises about 150 mg S-equol.
 7. The method ofclaim 1, wherein the formulation is administered orally, intravenously,intraperitoneally, or subcutaneously.
 8. The method of claim 1, whereinsaid subject is a human.
 9. The method of claim 1, wherein the S-equolis administered in combination with one or more other cancer treatments.10. The method of claim 9, wherein the S-equol is administered incombination with an immunotherapeutic agent.
 11. The method of claim 10,wherein the immunotherapeutic agent is an antibody.
 12. The method ofclaim 11, wherein the antibody is directed to programmed cell deathprotein 1 (PD-1).
 13. The method of claim 12, wherein the antibody isdirected to programmed death ligand 1 (PDL-1).
 14. The method of claim12, wherein the antibody is pembrolizumab.
 15. The method of claim 13,wherein the antibody is atezolizumab.
 16. The method of claim 13,wherein the antibody is avelumab.
 17. The method of claim 1, wherein theformulation is essentially free of genistein, daidzein, and/orIBSO03569.
 18. The method of claim 1, wherein genistein, daidzein,and/or IBSO03569 are not co-administered with S-equol.
 19. The method ofclaim 1, wherein the formulation is essentially free of R-equol.
 20. Themethod of claim 1, wherein the S-equol is produced chemically.